| 摘要 |
FIELD: biotechnology, medicine, endocrinology, hormones. ^ SUBSTANCE: invention relates to a method for preparing genetic-engineering human insulin for in preparing medicinal preparations used in treatment of diabetes mellitus. Method is carried out by culturing the strain-produce of fusion protein containing human proinsulin - Escherichia coli BL21/pPINS07(BL07) or Escherichia coli JM109/pPINS07, disruption of cells by disintegration, separation of body inclusions comprising a fusion protein. Then method involves preliminary washing out body inclusions, simultaneous dissolving protein and reduction of disulfide bonds in buffer with 5 mM of dithiothreitol and 1 mM of EDTA followed by renaturation and purification of the renaturated fusion protein by ion-exchange chromatography. Cleavage of fusion protein is carried out by the combined hydrolysis with trypsin and carboxypeptidase B in the mass ratio fusion protein, trypsin and carboxypeptidase B = 400:0.6:0.9, respectively. Purification of insulin is carried out by hydrophobic chromatography of reversed-phase high-performance liquid chromatography followed by gel-filtration, and insulin is isolated by crystallization in the presence of zinc salts. Invention provides decreasing the process for preparing genetic-engineering human insulin and to increase its yield. ^ EFFECT: improved preparing method of insulin. ^ 2 ex |
